1. Field of the Invention
This invention relates in general to the methods and systems for analyzing the blood samples using a particle analyzer, and more particularly to determining reticulocytes and hard-to-ghost cells.
2. Background
Each year, millions of Americans are affected by blood diseases. Examples of blood pathologies include various hematological malignancies, such as leukemias and lymphomas, hemoglobinopathies, which encompass a number of anemias of genetic origin, such as thalassemia, Cooley's Disease, and sickle-cell anemia (HbS disease), as well as various clotting and bleeding disorders. Blood abnormality can also be a secondary consequence associated with other conditions, such HIV/AIDS, malignant tumors, and autoimmune disorders. Most of these conditions have significant morbidity and mortality and commonly cause severe pain in the affected patient. Early diagnosis of these disorders is critical so patients who have the disease can receive proper treatment and disease management.
Blood is a specialized bodily fluid that delivers necessary substances, such as nutrients and oxygen, to the body's cells and transports waste products away from those same cells. The predominant cell in blood is the erythrocyte, i.e., red blood cell or red cell. In a peripheral blood smear, erythrocytes derive their reddish color from protein hemoglobin, and usually appear round or oval with a pale-staining center region. Their biconcave morphology increases the cell's surface area and facilitates diffusion of oxygen and carbon dioxide from the cell. A typical erythrocyte has a lifespan of about 120 days.
Erythrocytes develop from nucleated precursor cells in the bone marrow. Immature erythrocytes, i.e., reticulocytes, have organelles that contribute to an increased hemoglobin content and gas-carrying capacity. Reticulocytes can be recognized in peripheral blood smears when a special stain is used to stain their polyribosome or ribonucleic acid (RNA). Under typical conditions, reticulocytes account for about 1-2% of red blood cells in a sample. However, during certain periods of physical need, the reticulocyte count may increase.
Blood tests can be used to determine physiological and biochemical states, such as disease, mineral content, drug effectiveness, and organ function. In confirming or helping to confirm the diagnosis of diseases such as, for example, various forms of anemia or acute internal hemorrhage, the determination of reticulocytes can be of critical importance.
Automated reticulocyte analysis can be done using a particle analyzer such as a flow cytometer or hematology analyzer. Example particle analyzers include, the UniCel® D×H 800 System from Beckman Coulter and XT-2000 from Sysmex Corporation. The preparation of a blood sample for cytometric flow or hematology analysis generally involves taking a whole blood sample and performing one or both the steps of incubating the sample of blood with a vital stain such as New Methylene Blue (NMB) and diluting the blood sample with a hypotonic acid that clears hemoglobin. The staining precipitates RNA within the erythrocytes. Diluting with a hypotonic acid clears hemoglobin, leaving the stained RNA within the cells. The process of removing hemoglobin is commonly referred to as “ghosting.” The blood sample, or portion of it, is then subjected to analysis in a flow cell of a particle analyzer. Typically, cells in a sheath fluid pass through a point in the flow cell, one by one, where they are interrogated by one or more beams of light. Several measurements are generated for each passing cell. The interrogation of a single cell is referred to as a cell event. Commonly recorded measurements per cell event include, forward light scatter, axial light loss, and fluorescence. Some particle analyzers also collect a direct current impedance (DC) measurement which is a measure of how much impedance is exerted by a cell. The DC measurement, which is obtained from applying the maximum current such that the cell membrane is not permeated and no current flows through the cell, is also known as Coulter volume or volume.